PROJECT SUMMARY Understanding how the nervous system extracts and encodes features of an organism?s environment is a major goal of sensory biology and systems neuroscience. In the vertebrate olfactory system, projection neurons of the main olfactory bulb receive synaptic input from the primary sensory neurons in the peripheral olfactory epithelium. These projection neurons ? comprising the mitral and tufted cells ? relay sensory information to multiple higher order brain centers to drive a spectrum of innate and learned behaviors. Previous studies indicate that olfactory bulb projection neurons are functionally if not genetically heterogeneous: the mitral and tufted cells are anatomically, morphologically and physiologically distinct; mitral cells can be distinguished based on their differential expression of ion channel subunits and intrinsic physiological properties; and projection neurons display different patterns of innervation in higher order olfactory centers implicated in innate and learned olfactory behaviors. One view based on this large body of evidence is that subtypes of projection neurons represent different features of the odorant stimulus and carry parallel streams of information to different olfactory centers to generate behaviors. While electrophysiological properties, expression of selected molecular markers and connectivity have been used to describe differences among projection neurons, current approaches have not yet yielded a systematic classification of olfactory bulb projection neuron diversity. This information is necessary for elucidating in a comprehensive manner the principles underlying olfactory coding as information is relayed and transformed by the population of olfactory bulb projection neurons. In the present application, we propose to apply single cell RNA profiling by deep sequencing (single cell RNA-Seq) to generate a taxonomy of olfactory bulb projection neurons. The single cell approach relies upon each cell type?s unique transcriptional signature to define itself and therefore requires no a priori knowledge of the nature or extent of diversity within the population. The taxonomy of projection neurons will be independently validated by RNA in situ hybridizations. We will further test the hypothesis that projection neurons innervating different brain regions are genetically distinct. Importantly, the identification of cell type-specific genes will allow genetic access to the newly identified cell types and the circuits in which they function. Thus we expect the proposed R21 project to enable the future development of powerful genetic tools for targeting the subcircuits mediating specific olfactory behaviors.